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Wallach, J, Colestock, T, Cicali, B, Elliott, SP, Kavanagh, PV, Adejare, A, Dempster, NM and Brandt, SD
Syntheses and analytical characterizations of N-alkyl-arylcyclohexylamines. http://researchonline.ljmu.ac.uk/id/eprint/3240/
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Wallach, J, Colestock, T, Cicali, B, Elliott, SP, Kavanagh, PV, Adejare, A, Dempster, NM and Brandt, SD (2015) Syntheses and analytical characterizations of N-alkyl-arylcyclohexylamines. Drug Testing and Analysis. ISSN 1942-7611
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http://researchonline.ljmu.ac.uk/ Drug Testing and Analysis
Syntheses and analyti cal characterizations of N -alkyl - arylcyclohexylamines
Journal:For Drug Peer Testing and Analysis Review Manuscript ID: DTA-15-0185.R1
Wiley - Manuscript type: Research Article
Date Submitted by the Author: n/a
Complete List of Authors: Wallach, Jason; University of the Sciences, Department of Pharmaceutical Sciences Colestock, Tristan; University of the Sciences, Department of Pharmaceutical Sciences Cicali, Brian; University of the Sciences, Department of Pharmaceutical Sciences Elliott, Simon; ROAR Rorensics, Kavanagh, Pierce; School of Medicine, Trinity Centre for Health Sciences, St. James Hospital, Department of Pharmacology and Therapeutics Adejare, Adeboye; University of the Sciences, Department of Pharmaceutical Sciences Dempster, Nicola; Liverpool John Moores University, School of Pharmacy and Biomolecular Sciences Brandt, Simon; School of Pharmacy & Biomolecular Sciences , Liverpool John Moores University
New psychoactive substances, Research chemicals, Phencyclidine, NMDA Keywords: receptors, Arylcyclohexylamines
The rise in new psychoactive substances that are available as ‘research chemicals’ (RCs) remains a significant forensic and legislative challenge. A number of arylcyclohexylamines have attracted attention as RCs and continued to be encountered, including 3-MeO-PCP, 3-MeO-PCE and 3- MeO-PCPr. These compounds are commonly perceived as ketamine-like dissociative substances and are believed to act predominantly via antagonism of the N-methyl-D-aspartate (NMDA) receptor. To aid in the identification of newly emerging substances the syntheses of fifteen N- alkyl-arylcyclohexylamines are described. Analytical characterizations were Abstract: performed via gas chromatography and high performance liquid chromatography coupled to multiple forms of mass spectrometry as well as nuclear magnetic resonance spectroscopy, ultraviolet diode array detection and infrared spectroscopy. The series consisted of the N-alkyl derivatives (N-methyl, N-ethyl, N-propyl) of phenyl-substituted and isomeric 2-, 3- and 4-methoxy phenylcyclohexylamines, as well as the N-alkyl derivatives obtained from a 3-methylphenyl and 2-thienyl moiety. In addition to the presentation of a range of previously unreported data, it was also found that positional isomers of aryl methoxyl-substituted arylcyclohexylamines were readily distinguishable under a variety of analytical conditions.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 2 of 34
1 2 3 Syntheses and analytical characterizations of N-alkyl- 4 5 arylcyclohexylamines 6 7 Jason Wallach a, Tristan Colestock a, Brian Cicali a, Simon P. Elliott b, Pierce 8 V. Kavanagh c, Adeboye Adejare a, Nicola M. Dempster d, Simon D. Brandt d,* 9 10 11 a 12 Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of 13 the Sciences, Philadelphia, PA 19104, USA 14 15 b ROAR Forensics, Malvern Hills Science Park, Geraldine Road, WR14 3SZ, UK 16 17 c Department of Pharmacology and Therapeutics, School of Medicine, Trinity Centre for 18 Health Sciences,For St. James Hospital,Peer Dublin 8, IrelanReviewd 19 20 d School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom 21 22 Street, Liverpool L3 3AF, UK 23 24 25 *Correspondence to: Simon D. Brandt, School of Pharmacy and Biomolecular Sciences, 26 Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK. E Mail: 27 [email protected] 28 29 Running title: Characterization of N alkyl arylcyclohexylamines 30
31 32 33 Abstract 34 35 The rise in new psychoactive substances that are available as ‘research chemicals’ 36 37 (RCs) remains a significant forensic and legislative challenge. A number of 38 arylcyclohexylamines have attracted attention as RCs and continued to be 39 encountered, including 3 MeO PCP, 3 MeO PCE and 3 MeO PCPr. These 40 compounds are commonly perceived as ketamine like dissociative substances and 41 are believed to act predominantly via antagonism of the N methyl D aspartate 42 43 (NMDA) receptor. To aid in the identification of newly emerging substances the 44 syntheses of fifteen N alkyl arylcyclohexylamines are described. Analytical 45 characterizations were performed via gas chromatography and high performance 46 liquid chromatography coupled to multiple forms of mass spectrometry as well as 47 nuclear magnetic resonance spectroscopy, ultraviolet diode array detection and 48 infrared spectroscopy. The series consisted of the N alkyl derivatives ( N methyl, N 49 50 ethyl, N propyl) of phenyl substituted and isomeric 2 , 3 and 4 methoxy 51 phenylcyclohexylamines, as well as the N alkyl derivatives obtained from a 3 52 methylphenyl and 2 thienyl moiety. In addition to the presentation of a range of 53 previously unreported data, it was also found that positional isomers of aryl methoxyl 54 substituted arylcyclohexylamines were readily distinguishable under a variety of 55 analytical conditions. 56 57 58 59 60 http://mc.manuscriptcentral.com/dta 1 Page 3 of 34 Drug Testing and Analysis
1 2 3 Keywords: New psychoactive substances; ‘research chemicals’; phencyclidine; 4 NMDA receptors; arylcyclohexylamines 5 6 7 Introduction 8 9 The non medical use of dissociative drugs including 1 (1 phenylcyclohexyl)piperidine 10 (PCP), ketamine and their derivatives is a challenge to policy makers, clinicians and 11 forensic investigators charged with their identification. More recent examples of 12 13 dissociative drugs include ‘research chemicals’ (RCs) such as 3 MeO PCP, [1 4] 14 methoxetamine (MXE), diphenidine and 2 methoxydiphenidine (2 MXP). In the UK, 15 a range of arylcyclohexylamines were placed under control in 2013 following generic 16 legislation [5] but control status varies across the globe. Within one week of 17 introducing generic control, diphenidine was available for purchase in the UK, which 18 For Peer Review 19 added to the existing product catalogues of substances suspected to show [1] 20 dissociative properties in humans. The association of some those newly emerging 21 substances with acute toxicity [6 8] serves as a reminder that the accurate identification 22 of these substances is essential for the monitoring of new psychoactive 23 substances.[9,10] 24 25 26 The major pharmacological mechanism that appears to mediate a significant portion 27 of the therapeutically relevant and psychoactive effects of dissociative substances 28 includes uncompetitive antagonism of the N methyl D aspartate (NMDA) receptor. 29 NMDA receptor antagonists exemplify an important pharmacological class that 30 provides promising pharmacological tools. They are used or are being investigated in 31 a number of therapeutic areas including general anesthetics, neuroprotection, 32 [1,11,12] 33 management of neuropathic pain and depression. Issues linked with clinical 34 tolerability remain challenging for clinical development of this promising 35 pharmacological class.[1,13,14] 36 37 Recently a study of the receptor binding profiles of a series of dissociative legal highs 38 included 3 methoxyeticyclidine (3 MeO PCE) ( 3b ).[15] Consistent with reports of its 39 [1] 3 40 potent dissociative effects in humans it showed high affinity for (+) [ H]MK 801 41 labeled NMDA receptors (61 nM). In addition, affinity for the serotonin transporter 42 (115 nM) and lesser affinity at sigma 1 and sigma 2 receptor sites were observed. [15] 43 It is unknown how these additional pharmacological interactions may contribute to 44 the psychoactive properties of 3 MeO PCE and related arylcyclohexylamines and 45 46 further studies are warranted to explore polypharmacological mechanisms that may 47 be relevant as well. 48 49 N Alkyl arylcyclohexylamines including PCE, PCPr and PCiP (Figure 1) were sold on 50 ‘street markets’ during the 1960 1990s in the United States and PCE in particular 51 enjoyed a large distribution relative to other PCP derivatives. [1] Furthermore, a 52 53 number of N alkoxyalkyl secondary amine derivatives were detected in Germany 54 during the 1990s and included PCMEA, PCEEA, PCMPA and PCEPA (Figure 1), 55 respectively.[16 18] More recently, 3 MeO PCE ( 3b )[19] and 3 MeO PCPr (3c) have 56 been sold through Internet based vendors (Figure 1).[1,20] Several closely related 57 compounds included derivatives of ketamine, such as methoxetamine, 2 MeO 2 58 59 60 http://mc.manuscriptcentral.com/dta 2 Drug Testing and Analysis Page 4 of 34
1 2 3 deschloroketamine (‘2 MeO ketamine’, 2 MK) and N ethylnorketamine (‘ N ethyl 4 ketamine’, N EK) (Figure 1) were previously reported.[1,19,21] The analytical 5 characterizations of a series of tertiary amine based arylcyclohexylamines including 6 ‘research chemicals’ 3 MeO and 4 MeO PCP was previously reported.[4] Although a 7 [20] 8 previous publication described the appearance of 3 MeO PCE, detailed 9 information on analytical profiles of the secondary amine N alkyl 10 arylcyclohexylamines are lacking. 11 12 This study presents the syntheses of 15 N alkyl arylcyclohexylamines (1a ) – (5c ) 13 using facile routes that are easily adaptable to forensic laboratories. The series 14 15 included the N alkyl derivatives ( N methyl, N ethyl, N propyl) of 2 , 3 and 4 MeO 16 arylcyclohexylamine isomers as well as 3 methylphenyl and 2 thienyl compounds 17 (Figure 2A). The comprehensive analytical characterizations included the 18 differentiation For between Peer positional isomersReview of aryl methoxyl substituted 19 arylcyclohexylamines. 20 21 22 23 Experimental 24 25 Materials 26 27 28 All starting materials, reagents and solvents used for synthesis (≥ 96%) were 29 obtained from Sigma Aldrich (St. Louis, USA). Column chromatography was 30 conducted using Merck silica gel, grade 9385 (230 400 mesh, 60 Å). Melting point 31 ranges were obtained using a DigiMelt A160 SRS melting point apparatus (Stanford 32 Research Systems, Sunnyvale, USA) at a ramp rate of 2 °C/min. 33 34 35 Synthesis procedures 36 A representative example is shown for the preparations of (1a ) – (1c ), where the 37 appropriately substituted Grignard reagent served as the starting point (Figure 2B). 38 NMR data for compounds (1a ) – (5c) are provided in Tables 1–6. NMR data of the 39 primary amines are shown as supplementary information. 40 41 42 Preparation of 1-(thiophen-2-yl)cyclohexan-1-amine (TCA) 43 44 A solution containing the desired Grignard reagent was prepared consisting of 2 45 bromothiophene (147 mmol, 24.08 g) in 200 mL dry THF containing freshly crushed 46 Mg (442 mmol, 10.75 g) at room temperature under argon. After stirring for 12 hours 47 48 at room temperature, cyclohexanone (114 mmol, 11.14 g) was added slowly. The 49 exothermic reaction mixture was then stirred for an additional 24 hours at which point
50 it was quenched with 300 mL distilled water (dH 2O), titrated to pH 7 with a saturated 51 NH 4Cl solution and extracted with ethyl acetate (3 x 100 mL). Organic phases were 52 pooled, washed with a saturated sodium bisulfite solution (2 x 150 mL), saline (1 x 53 54 150 mL), dried with anhydrous sodium sulfate and concentrated under reduced 55 pressure to give 1 (thiophene 2 yl)cyclohexane 1 ol as an amber oil. 56
57 A solution of the crude tertiary alcohol (104 mmol, 18.94 g) in CHCl 3 (50 mL) was 58 added dropwise to a vigorously stirred suspension of NaN 3 (208 mmol, 13.51 g) and 59 60 http://mc.manuscriptcentral.com/dta 3 Page 5 of 34 Drug Testing and Analysis
1 2 3 trifluoroacetic acid (312 mmol, 23.86 mL) in CHCl 3 (200 mL) at 0 °C under argon. 4 After addition, the suspension was allowed to recover to room temperature and stir 5 for 24 hours at which point it had set to a solid mass. The reaction mixture was 6 carefully quenched by slow addition to a stirred concentrated aqueous solution of 7 8 NaHCO 3. Once the evolution of gas was complete, the organic phase was collected, 9 washed with dH 2O and saline, dried with anhydrous sodium sulfate and concentrated 10 under reduced pressure to give the crude azide as a yellow oil. The azide was 11 purified using flash column chromatography on silica gel with hexanes as the mobile 12 phase. Early fractions containing the azide product were pooled and concentrated to 13 produce a light yellow oil (81 mmol, 16.71 g, 77.9% yield). 14 15 16 A solution of the purified azide (81 mmol, 16.71 g) in dry THF (50 mL) was added
17 dropwise over the course of an hour to a vigorously stirred suspension of LiAlH 4 (242 18 mmol, 9.2 g) inFor dry THF (200 Peer mL) under argon Review at 0°C. The reaction mixture was kept 19 at 0 °C for 1 h. It was then quenched by cautious dropwise addition of a 50:50 20 21 mixture of THF and ice cold dH2O (50 mL). A few mL of aqueous KOH were added to 22 ensure a basic pH and ethyl acetate (300 mL) was added and the suspension gravity 23 filtered to remove insoluble inorganic salts. Inorganic salts were washed with ethyl 24 acetate. The organic phase was extracted with aqueous HCl solution (3 x 200 mL), 25 acidic phases pooled, made basic to pH > 12 with KOH pellets and extracted with 26 ethyl acetate (3 x 70 mL). Organic phases were pooled, washed with saline, dried 27 28 with anhydrous magnesium sulfate, gravity filtered and concentrated under reduced 29 pressure to give a colorless oil. The crude product was purified by flash column 30 chromatography on silica gel eluting with hexanes:ethyl acetate (3:1) containing 31 0.5 % triethylamine with a gradually increasing ethyl acetate concentration to 50%. 32 Desired fractions were pooled and concentrated under reduced pressure to give 1 33 (thiophene 2 yl)cyclohexane 1 amine (TCA), a light yellow oil (71 mmol, 12.85 g, 34 35 87.7% yield). 36 37 The HCl salt of TCA was prepared by dissolving it in ethanol, titrating to pH 1.0 with 38 concentrated HCl, and evaporating under a stream of warm air. Dry acetone (4 Å 39 sieves) was added in 10 mL increments until all residual moisture and HCl was 40 41 removed. The resulting solid was washed with ethyl acetate (2 x 5 mL), dried, and 42 crystallized by dissolving in a minimal amount of warm methanol diluted with a 10 43 fold excess of diethyl ether. The solution was stored at 0 oC overnight. The resulting 44 crystals were collected by decanting the solvent off and then washing with ethyl 45 acetate (2 x 5 mL), followed by drying in an oven at 60 oC. The solids were 46 recrystallized two more times as described to produce white fluffy thin needle like 47 48 crystalline solids (m.p. 214.3 – 215.6 °C). 49 50 Preparation of N-methyl-1-(thiophen-2-yl)cyclohexan-1-amine (TCMe) (1a ) 51 52 A solution of TCA (7.72 mmol, 1.40 g) and ethyl formate (10 mL) containing 4 Å 53 molecular sieves (1.0 g) were refluxed under an inert atmosphere of argon. As 54 55 solvent tended to dissipate, additional ethyl formate was added as needed to keep a 56 constant volume. After ~72 h the reaction mixture was quenched by diluting in 400 57 mL 1N aqueous HCl solution. This solution was extracted with ethyl acetate (3 x 60 58 mL), organic phases pooled, washed with 2N aqueous HCl solution (2 x 100 mL), 59 60 http://mc.manuscriptcentral.com/dta 4 Drug Testing and Analysis Page 6 of 34
1 2 3 saline (40 mL), dried and concentrated under vacuum to give N [1 (thiophen 2 4 yl)cyclohexyl]formamide ( N formyl TCA) as a white solid (6.69 mmol, 1.40 g, 86.7 % 5 yield). The solid was crystallized from hexanes containing 10% ethyl acetate followed 6 by storage at 0 °C. However, the crude product was sufficient for use in the 7 8 subsequent reaction. 9 10 A solution of dry THF (40 mL) containing N formyl TCA (6.69 mmol, 1.40 g) was 11 added dropwise to a stirred suspension of LiAlH 4 (21.4 mmol, 0.8117 g) in THF (100 12 mL) at 0 °C under argon. The stirred reaction mixture was then allowed to warm to 13 room temperature and placed on a mild reflux while maintaining an inert argon 14 15 atmosphere. Once complete (TLC or GC/MS), the reaction mixture was placed on ice 16 and quenched by dropwise addition of a 50:50 mixture of ice cold THF and dH 2O with 17 vigorous stirring. The suspension was then diluted with 400 mL ethyl acetate and the 18 inorganic saltsFor removed byPeer gravity filtration. Review The filtered inorganic material was 19 washed heavily with ethyl acetate, and the washings combined and extracted with 20 21 aqueous 1N HCl (3 x 200 mL). The pooled aqueous phases were then basified to 22 >pH 12 with KOH pellets and extracted with ethyl acetate (3 x 60 mL). The pooled 23 organic extractions were washed with saline, dried with anhydrous magnesium 24 sulfate and concentrated under reduced pressure to give an amber oil. The crude 25 product was purified using flash column chromatography on silica gel eluting with 26 hexanes:ethyl acetate (4:1) containing 1% triethylamine. Desired fractions were 27 28 pooled and concentrated yielding colorless TCMe oil (4.92 mmol, 960 mg, 73.5 % 29 yield). HCl salt prepared as described to give a colorless needle like crystalline solid 30 (148 − 149 °C (phase transition), m.p. 194.8 − 195.9 °C). HR ESI MS: observed m/z 31 + + 196.11553 (theory [M + H] : C11 H18 NS m/z 196.11545). 32 33 1b 34 Preparation of N-ethyl-1-(thiophen-2-yl)cyclohexane-1-amine (TCE) ( ) 35 36 TCA (5.52 mmol, 1.0 g) was dissolved in triethylamine (1 mL) in a beaker and placed 37 on ice. Under a blanket of argon gas, acetyl chloride (11.1 mmol, 0.79 mL) was 38 added dropwise and mixed with a spatula until a waxy white precipitate formed. The 39 solid was suspended in aqueous 2N HCl solution (100 mL) and extracted with ethyl 40 41 acetate (3 x 75 mL). The pooled organic phases were washed with 2N aqueous HCl 42 solution (2 x 30 mL), dH 2O (20 mL) and saline (10 mL). The organic solution was 43 dried with anhydrous sodium sulfate and concentrated under reduced pressure to 44 produce N [1 (thiophene 2 yl)cyclohexyl]acetamide (4.92 mmol, 1.10 g, 89.5 % yield). 45 These crystals were recrystallized from boiling ethyl acetate and stored at 0 °C. The 46 crude product was sufficient in purity for use in the subsequent reaction. 47 48 49 A solution of N [1 (thiophene 2 yl)cyclohexyl]acetamide (4.5 mmol, 1.0 g) in dry THF 50 (20 mL) was added dropwise to a stirred suspension of LiAlH 4 (13.4 mmol, 0.51 g) in 51 dry THF (100 mL) on ice under argon. Upon completion of the addition, the reaction 52 mixture was removed from ice and brought to a mild reflux. The reflux was continued 53 and monitored for completion by TLC (~ 4 h). The reaction mixture was quenched 54 55 with dropwise addition of a 50:50 mixture of ice cold THF and dH 2O. A few drops of 56 KOH solution were added and the reaction mixture diluted with ethyl acetate (200 57 mL). The resulting inorganic salts were removed via gravity filtration and washed with 58 ethyl acetate (150 mL). The organic phase was extracted with aqueous 1N HCl 59 60 http://mc.manuscriptcentral.com/dta 5 Page 7 of 34 Drug Testing and Analysis
1 2 3 solution (3 x 75 mL). The pooled aqueous phases were made basic with KOH pellets 4 (pH >12) and extracted with ethyl acetate (3 x 100 mL). The pooled organic fractions 5 were washed with saline (20 mL), dried with anhydrous magnesium sulfate, gravity 6 filtered and concentrated under reduced pressure to produce N ethyl 1 (thiophene 2 7 8 yl)cyclohexane 1 amine (1b ) (2.10 mmol, 0.45 g, 46.7 % yield). HCl salt prepared as 9 previously described to give a colorless needle like crystalline solid (m.p. 193.9 – 10 195.1 °C; lit: 195 196 °C [22] ) HR ESI MS: observed m/z 210.13120 (theory [M + H] +: + 11 C12 H20 NS m/z 210.13110). 12 13 Preparation of N-propyl-1-(thiophen-2-yl)cyclohexane-1-amine (TCPr) (1c ) 14 15 16 TCPr was prepared as described for TCE above in 92% yield from TCA (5.52 mmol, 17 1.0 g) as a white crystalline solid. Generation of the HCl salt gave a colorless needle 18 like crystalline Forsolid (116 −Peer 117 °C (phase Review transition), m.p. 183.8 − 185.5 °C). HR 19 ESI MS: observed m/ (theory [M + H] +: C H NS + m/z 224.1467). 20 z 224.1465 13 22 21 22 Preparation of 1-(2-methoxyphenyl)cyclohexan-1-amine (2-MeO-PCA) 23 24 2 MeO PCA was prepared in 54.8% yield from cyclohexanone (26.6 mmol, 2.61 g) 25 and 2 bromoanisole (31.9 mmol, 5.96 g) as described for TCA above. The HCl salt 26 27 was prepared as described for 3 MeO PCA to give colorless needles with a melting 28 point of 176.7 − 178.0 °C (lit: 212 215 °C [23] ). 29 30 Preparation of N-methyl-1-(2-methoxyphenyl)cyclohexan-1-amine (2-MeO-PCMe) 31 (2a) 32 33 34 N [1 (2 methoxyphenyl)cyclohexyl]formamide ( N formyl 2 MeO PCA) was prepared 35 as previously described in 67.7 % yield from 2 MeO PCA (5.21 mmol, 1.07 g) as a 36 white solid. 2 MeO PCMe was obtained as described in 16.9% yield from N formyl 2 37 MeO PCA (0.86 mmol, 0.20 g), as a colorless oil. The HCl salt was obtained as a 38 white crystalline solid; 107 − 110 °C (phase transition), m.p. 185 − 187 °C. HR ESI 39 + + 40 MS: observed m/z 220.16982 (theory [M + H] : C14 H22 NO m/z 220.16959). 41 42 Preparation of N-ethyl-1-(2-methoxyphenyl)cyclohexan-1-amine (2-MeO-PCE) (2b ) 43 44 N [1 (2 methoxyphenyl)cyclohexyl]acetamide ( N acetyl 2 MeO PCA) was prepared 45 46 as previously described in 93.6 % yield from 2 MeO PCA (5.50 mmol, 1.13 g) as a 47 white solid. 2 MeO PCE freebase was prepared as described in 91.2% yield from N 48 acetyl 2 MeO PCA (3.92 mmol, 0.97 g) as a colorless oil. The sparkling white 49 crystalline HCl salt gave a m.p. of 194.3 − 195.9 °C. HR ESI MS: observed m/z 50 234.18520 (theory [M + H] +: C H NO + m/z 234.18524). 51 15 24
52 2c 53 Preparation of N-propyl-1-(2-methoxyphenyl)cyclohexan-1-amine (2-MeO-PCPr) ( ) 54 55 N [1 (2 methoxyphenyl)cyclohexyl]propanamide ( N propionyl 2 MeO PCA) was 56 prepared as previously described in 88.0 % yield from 2 MeO PCA (5.41 mmol, 1.11 57 g) as a white solid. 2 MeO PCPr was prepared as described in 28.9% yield from N 58 59 60 http://mc.manuscriptcentral.com/dta 6 Drug Testing and Analysis Page 8 of 34
1 2 3 propionyl 2 MeO PCA (1.34 mmol, 0.35 g), as a colorless oil. The HCl salt was a 4 sparkling white crystalline solid (m.p. 213.5 − 214.5 °C). HR ESI MS: observed m/z 5 248.20086 (theory [M + H] +: C H NO + m/z 248.20089). 6 16 26 7 8 Preparation of 1-(3-methoxyphenyl)cyclohexan-1-amine (3-MeO-PCA) 9 10 3 MeO PCA was prepared in 61.0% yield from cyclohexanone (53.0 mmol, 5.23 g) 11 and 3 bromoanisole (64.0 mmol, 11.97 g). The HCl salt of 3 MeO PCA was prepared 12 as described previously (m.p. 197.0 − 197.7 °C [4] ; lit: 195 − 196 °C [23] ). 13 14 15 Preparation of N-methyl-1-(3-methoxyphenyl)cyclohexan-1-amine (3-MeO-PCMe) 16 (3a ) 17 18 N [1 (3 methoxyphenyl)cyclohexyl]formamideFor Peer Review ( N formyl 3 MeO PCA) was prepared 19 20 as previously described in 97.0% yield from 3 MeO PCA (10.2 mmol, 2.10 g) as a 21 white solid. 3 MeO PCMe was prepared as described in 79.0% yield from N formyl 3 22 MeO PCA (9.94 mmol, 2.32 g), as colorless oil. The HCl salt of 3 MeO PCMe (3a ) 23 was prepared and recrystallized twice from methanol/diethyl ether at 0 °C to give a 24 colorless, crystalline solid (m.p. 214.5 − 217.0 °C). HR ESI MS: observed m/z 25 220.16948 (theory [M + H] +: C H NO + m/z 220.16959). 26 14 22 27 28 Preparation of N-ethyl-1-(3-methoxyphenyl)cyclohexan-1-amine (3-MeO-PCE) (3b ) 29 30 N [1 (3 methoxyphenyl)cyclohexyl]acetamide ( N acetyl 3 MeO PCA) was prepared 31 as previously described in 99.1% yield from 3 MeO PCA (6.33 mmol, 1.30 g) as a 32 deep orange oil. 3 MeO PCE prepared in 55.3% yield as described from N acetyl 3 33 34 MeO PCA (1.82 mmol, 0.45 g). HCl salt was prepared to give a white crystalline solid, 35 m.p. 214.0 − 215.5 °C. HR ESI MS: observed m/z 234.18519 (theory [M + H] +: + 36 C15 H24 NO m/z 234.18524). 37 38 Preparation of N-propyl-1-(3-methoxyphenyl)cyclohexan-1-amine (3-MeO-PCPr) (3c ) 39 40 41 N [1 (3 methoxyphenyl)cyclohexyl]propanamide ( N propionyl 3 MeO PCA) was 42 prepared as previously described in 91.0% yield from 3 MeO PCA (6.33 mmol, 1.30 43 g) as an orange colored oil. 3 MeO PCPr prepared as described in 75.5% yield from 44 N propionyl 3 MeO PCA (3.82 mmol, 1.0 g) as a colorless oil. HCl salt was prepared 45 46 as described to give a white crystalline solid, m.p. 181.0 − 183.0 °C. HR ESI MS: + + 47 observed m/z 248.20063 (theory [M + H] : C16 H26 NO m/z 248.20089). 48 49 Preparation of 1-(4-methoxyphenyl)cyclohexan-1-amine (4-MeO-PCA) 50 51 4 MeO PCA was prepared as previously described in 49.0% yield from 52 53 cyclohexanone (41.5 mmol, 4.07 g) and 4 bromoanisole (49.7 mmol, 9.30 g) as 54 colorless oil. HCl salt was prepared to give a thin needle like crystalline solid, m.p. 55 251.1 − 252.0 °C (Lit: 233 − 234 °C [23] ) 56 57 58 59 60 http://mc.manuscriptcentral.com/dta 7 Page 9 of 34 Drug Testing and Analysis
1 2 3 Preparation of N-methyl-1-(4-methoxyphenyl)cyclohexan-1-amine (4-MeO-PCMe) 4 (4a) 5 6 N [1 (4 methoxyphenyl)cyclohexyl]formamide ( N formyl 4 MeO PCA) was prepared 7 8 as previously described in 87.0 % yield from 4 MeO PCA (5.16 mmol, 1.06 g) as a 9 white solid. 4 MeO PCMe was prepared in 39.5% yield from N formyl 4 MeO PCA 10 (3.34 mmol, 0.780 g), as a colorless oil. HCl salt was prepared as described to give a 11 sparkling colorless needle like solid, m.p. 164.0 − 165.7 °C. HR ESI MS: observed 12 m/z 220.1964 (theory [M + H] +: C H NO + m/z 220.1696). 13 14 22 14 15 Preparation of N-ethyl-1-(4-methoxyphenyl)cyclohexan-1-amine (4-MeO-PCE) (4b ) 16 17 N [1 (4 methoxyphenyl)cyclohexyl]acetamide ( N acetyl 4 MeO PCA) was prepared 18 as previously describedFor in Peer90.5 % yield from Review 4 MeO PCA (6.92 mmol, 1.42 g) as a 19 white solid. 4 MeO PCE was prepared as described in 50.8% yield from N acetyl 4 20 21 MeO PCA (2.42 mmol, 0.60 g), as a colorless oil. HCl salt was prepared as 22 described to give a sparkling white crystalline solid, m.p. 189.0 − 191.8 °C. HR ESI + + 23 MS: observed m/z 234.1850 (theory [M + H] : C15 H24 NO m/z 234.1852). 24 25 Preparation of N-propyl-1-(4-methoxyphenyl)cyclohexan-1-amine (4-MeO-PCPr) (4c ) 26 27 28 N [1 (4 methoxyphenyl)cyclohexyl]propanamide ( N propyl 4 MeO PCA) was 29 prepared as previously described in 86.0 % yield from 4 MeO PCA (6.82 mmol, 1.40 30 g) as a white solid. 4 MeO PCPr was prepared as described in 39.4% yield from N 31 proply 4 MeO PCA (2.30 mmol, 0.60 g), as a colorless oil. HCl salt was prepared as 32 described to give a sparkling white crystalline solid, m.p. 184.8 − 186.3 °C. HR ESI 33 + + 34 MS: observed m/z 248.2006 (theory [M + H] : C16 H26 NO m/z 248.2009). 35 36 Preparation of 1-(3-methylphenyl)cyclohexan-1-amine (3-Me-PCA) 37 38 3 Me PCA was prepared as described in 70.2% yield from cyclohexanone (25.0 39 40 mmol, 2.45 g) and 3 bromotoluene (30.0 mmol, 5.13 g) as colorless oil. HCl salt was 41 prepared to give a white fluffy crystalline solid, m.p. 209 − 212 °C (Lit: 209 42 − 210 °C [23] ). 43 44 Preparation of N-methyl-1-(3-methylphenyl)cyclohexan-1-amine (3-Me-PCMe) (5a) 45 46 47 N [1 (3 methylphenyl)cyclohexyl]formamide (N formyl 3 Me PCA) was prepared as 48 previously described in 95.9% yield from 3 Me PCA (11.1 mmol, 2.1 g) as a light 49 yellow oil. 3 Me PCMe was prepared as described in 59.0% yield from N formyl 3 50 Me PCA (10.6 mmol, 2.31 g), as a colorless oil. HCl salt was prepared to give fluffy 51 52 colorless needle like crystalline solid, m.p. 213.3 − 215.5 °C. HR ESI MS: observed + + 53 m/z 204.17439 (theory [M + H] : C14 H22 N m/z 204.17468). 54 55 Preparation of N-ethyl-1-(3-methylphenyl)cyclohexan-1-amine (3-Me-PCE) (5b ) 56 57 58 59 60 http://mc.manuscriptcentral.com/dta 8 Drug Testing and Analysis Page 10 of 34
1 2 3 N [1 (3 methylphenyl)cyclohexyl]acetamide ( N acetyl 3 Me PCA) was prepared as 4 described in 95.5% yield from 3 Me PCA (5.28 mmol, 1.0 g) as an orange crystalline 5 solid. 3 Me PCE prepared as described in 76.4% yield from N acetyl 3 Me PCA 6 (6.48 mmol, 1.50 g), as a colorless oil. HCl salt was prepared to give a white 7 [24] 8 crystalline solid, m.p. 234.1 − 235.0 °C (Lit: 236 − 237 °C ). HR ESI MS: observed + + 9 m/z 218.19030 (theory [M + H] : C15 H24 N m/z 218.19033). 10 11 Preparation of N-propyl-1-(3-methylphenyl)cyclohexan-1-amine (3-Me-PCPr) (5c ) 12 13 14 N [1 (3 methylphenyl)cyclohexyl]propanamide ( N propionyl 3 Me PCA) was 15 prepared as previously described in 86.5 % yield from 3 Me PCA (5.28 mmol, 1.0 g) 16 as a white solid. 3 Me PCPr was prepared as described in 65.0% yield from N 17 propionyl 3 Me PCA (5.30 mmol, 1.30 g), as a colorless oil. HCl salt was prepared to 18 give a fluffy whiteFor crystalline Peer solid, m.p. 226.0 Review − 226.8 °C. HR ESI MS: observed m/z 19 + + 20 232.20588 (theory [M + H] : C16 H26 N m/z 232.20598). 21 22 Instrumentation 23 24 Nuclear magnetic resonance spectroscopy 25 26 1 13 27 H (400 MHz) and C NMR spectra (100 MHz) were obtained from the hydrochloride 28 salts in CDCl 3 solutions (100% and 99.96% D, 0.03% (v/v) TMS) on a Bruker 29 Ultrashield 400 plus spectrometer with a 5 mm BBO S1 (Z gradient plus) probe at 30 24 °C. Internal chemical shift references were TMS (δ = 0.00 ppm) and solvent (δ = 31 77.0 ppm). 32 33 34 Gas chromatography ion trap mass spectrometry 35 36 Data for all fifteen N alkyl arylcyclohexylamines (0.5 mg/mL in methanol) were 37 recorded under full can electron (EI) and chemical ionization (CI) conditions using 38 39 HPLC grade methanol as the liquid CI reagent. A Varian 450 GC gas chromatograph 40 coupled to a Varian 220 MS ion trap mass spectrometer and a Varian 8400 41 autosampler was employed with a Varian CP 1177 injector (275 ºC) in split mode 42 (1:50) (Walnut Creek, CA, USA). The Varian MS Data Review function of the 43 Workstation software, version 6.91, was used for data acquisition. Transfer line, 44 manifold and ion trap temperatures were set at 310, 80 and 220 ºC, respectively. The 45 46 carrier gas was helium at a flow rate of 1 mL/min using the EFC constant flow mode. 47 The default settings for CI ionization parameters (0.4 s/scan) were used: CI storage 48 level m/z 19.0; ejection amplitude m/z 15.0; background mass m/z 55; maximum 49 ionization time 2000 s; maximum reaction time 40 ms; target TIC 5000 counts. An 50 Agilent J&W VF 5ms GC column (30 m × 0.25 mm, 0.25 m) was employed for 51 52 separation. The starting temperature was set at 130 ºC and held for 1 min. The 53 temperature then increased at 20 ºC/min to 280 ºC and held constant for 11.50 min 54 to give a total run time of 20.00 min. 55 56 Electrospray triple quadrupole mass spectrometry 57 58 59 60 http://mc.manuscriptcentral.com/dta 9 Page 11 of 34 Drug Testing and Analysis
1 2 3 Electrospray triple quadrupole tandem mass spectrometry experiments were carried 4 out by direct infusion (10 L/min at 0.01 mg/mL) of ( 1a ) – (5c ) compounds using a 5 Waters Micromass Quattro Premier triple quadrupole MS/MS system (Waters 6 7 Micromass, Manchester, UK) and the Masslynx version 4.1 software. Optimization of 8 signal intensities were performed in positive MS scan and in product ion scan mode. 9 The optimized source conditions were as follows: capillary 3.12 kV, rf lens 0.1 V, 10 source temperature 100 °C, desolvation temperature 200 °C and the multiplier 11 voltage was 650 V. Nitrogen was used as the cone gas (50 L/h) and desolvation gas 12 (200 L/h) whilst the collision gas was argon (0.3 mL/min flow). The [M + H] + ion 13 14 corresponding to all fifteen substances were selected for MS/MS experiments. 15 Product ions were collected over the range m/z 50 and m/z 250. Scan time for each 16 channel was 0.5 s and interscan delay was 0.1 s. The cone voltage and collision 17 energy values were as follows: 10 V and 18 eV for (1a) – (1c); 18 V and 15 eV for all 18 three sets of methoxyphenylFor Peer substituted isomers;Review 15 V and 15 eV for (5a ) and (5c ) 19 and 15 V and 12 eV for (5b ), respectively. 20 21 22 23 High-resolution electrospray mass spectrometry 24 25 Analyses were carried out by characterization using UHPLC QTOF MS/MS as 26 described previously. [25,26] Briefly, mobile phases used for UHPLC separation 27 consisted of 100% acetonitrile (1% formic acid) and an aqueous solution of 1% 28 formic acid. The column temperature was set at 40 °C (0.6 mL/min) and data were 29 30 acquired for 5.5 min. The elution was a 5 –70% acetonitrile gradient ramp over 3.5 31 min, then increased to 95% acetonitrile in 1 min and held for 0.5 min before returning 32 to 5% acetonitrile in 0.5 min. QTOF MS data were acquired in positive mode 33 scanning from m/z 100 – m/z 1000 with and without auto MS/MS fragmentation. 34 Ionization was achieved with an Agilent JetStream electrospray source and infused 35 internal reference masses. Agilent 6540 QTOF MS parameters: gas temperature 36 37 325 °C, drying gas 10 L/min and sheath gas temperature 400 °C. Internal reference 38 ions at m/z 121.05087 and m/z 922.00979 were used. 39 40 High performance liquid chromatography diode array detection 41 42 HPLC DAD analyses [25,26] were carried out on a Dionex 3000 Ultimate system 43 44 coupled to a UV diode array detector (Thermo Fisher, St Albans, UK), using a 45 Phenomenex Synergi Fusion column (150 mm x 2 mm, 4 m) that was protected by 46 a 4 mm x 3 mm Phenomenex Synergi Fusion guard column (Phenomenex, 47 Macclesfield, UK). The mobile phases were made from 70% acetonitrile with 25 mM 48 TEAP buffer and an aqueous solution of 25 mM TEAP buffer. Elution was achieved 49 with a gradient that started with 4% acetonitrile and ramped to 70% acetonitrile in 15 50 51 min and held for 3 min. The total acquisition time was 18 min at a flow rate of 0.6 52 mL/min. The diode array detection window was set at 200 nm to 595 nm (collection 53 rate 2 Hz). 54 55 Infrared spectroscopy 56
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1 2 3 Infrared (IR) spectra were obtained on a Perkin Elmer Spectrum BX FTIR model 4 (Llantrisant, UK) using a Pike MIRacle ATR system. Data were acquired with the 5 Spectrum v5.01 software (scan range 4000 –400 cm 1, resolution 4 cm 1, 16 scans). 6
7 8 Results and discussion 9 10 A number of routes exist for the syntheses of arylcyclohexylamines. [1,27] In the 11 present study, primary amines were synthesized using modified Geneste route 12 [28] 13 (Figure 2B) , similar to the approach taken in previous work on PCP and 1 (1 [4] 14 phenylcyclohexyl)pyrrolidine (PCPy) analogs. It has been found in practice that the 15 primary amines represent practical intermediates to prepare appropriately substituted 16 secondary and tertiary amines. Secondary amines were synthesized by conversion 17 to the amide, which was found to be high yielding. Subsequent reduction of the 18 For Peer Review 19 amide gave the desired N alkyl secondary amines (1a ) – (5c) in moderate to good 20 yields. 21 22 The syntheses of primary amine intermediates 3 Me PCA, 2 MeO PCA, 3 MeO PCA, 23 4 MeO PCA and TCA have been described previously, [22,23,29,30] and these were 24 based on the implementation of the Geneste route in most cases. A modified imine 25 [31] 26 (sulfinimine) route has been described for 3 MeO PCA and a route via an imine 27 stage was employed before for the preparation of secondary amine compounds 28 carrying a 3 methoxyphenyl substituent. [32] The preparation of secondary amines 29 from primary amine intermediates via alkylation with the appropriate alkyl halide was 30 also reported for TCMe ( 1a ), TCE ( 1b ) and TCPr ( 1c ). [30,33] The syntheses of the 31 methoxy isomers was described in 1988 using an imine route. [32] 32 33 34 The characterization of a number of compounds appeared to be unreported in the 35 existing literature, such as 2 MeO PCE (2b ), 2 MeO PCPr ( 2c ), 3 Me PCPr ( 5c ), and 36 4 MeO PCPr ( 4c ). Likewise, 2 MeO PCMe ( 2a ) and 4 MeO PCMe ( 4a ) do not 37 appear in the literature but are apparently available commercially. It was previously 38 suggested [1] that 3 MeO PCE ( 3b ), and 3 MeO PCPr ( 3c ) had not been described in 39 40 the scientific literature prior to their non medical use. However, it was noticed that 41 these were in fact described in a 1988 conference proceedings held by researchers 42 at Eli Lilly. Both compounds showed NMDA receptor affinity and in vivo activity 43 consistent with NMDA receptor antagonism. [32] 44 45 46 Chromatography and mass spectrometry 47 48 Gas chromatography retention times and ion trap (IT) electron (EI) and chemical 49 ionization (CI) mass spectral data (GC IT MS) are summarized in Figures 3 and 4. 50 The proposed mechanism for EI induced formation of base peak ions, which may 51 have involved the loss of a propyl radical from the cyclohexyl component, was 52 [4] 53 adapted from previously published work and is shown in Figure 5A . The EI and CI 54 mass spectra recorded for 3 MeO PCE ( 3b ) (Figure 3H1/H2) were comparable to 55 previously published data [20] but some differences in relative abundance values were 56 also visible, which might have been associated with the ion trap mass analyzer. For 57 example, under the conditions used, the implementation of GC ion trap EI MS 58 59 60 http://mc.manuscriptcentral.com/dta 11 Page 13 of 34 Drug Testing and Analysis
1 2 3 frequently resulted in the formation of what appeared to be the protonated molecule 4 instead of the M •+ ion. In some cases, formation of the [M + H] + ion formed the base 5 peak similar to what was observed previously during the analysis of methoxetamine 6 (MXE). [20,34] In the present study, it was noticed that [M + H] + ions were particularly 7 8 abundant in the EI IT MS spectra of the 2 methoxyphenyl substituted compounds 9 (2a) – (2c) (Figure 3D1, 3E1 and 3F1). Self ionization was not observed in any EI 10 mass spectra recorded with a quadrupole mass analyzer where M •+ ions were 11 detectable in appreciable abundance (supplementary information). 12 13 When inspecting the EI IT mass spectra for potential differences between N methyl 14 15 substituted isomers (2a, 3a and 4a ), it was observed that an ion at m/z 189 was 16 particularly abundant in the EI IT mass spectrum of 4 MeO PCMe ( 4a) (Figure 4J1) 17 and that it was absent when using a quadrupole mass analyzer (supplemental data). 18 Figure 5B showsFor a suggested Peer mechanism Review that may account for this observation 19 following the neutral loss of formaldehyde (CH O) within the ion trap. The ortho and 20 2 21 meta isomers (2a and 3a ) (Figure 3D1 and 3G1) indicated a reduced formation of 22 m/z 189 which may be explained by the proximity of the 2 OCH 3 group to the terminal 23 methylene radical. Interestingly, the isomers 2 , 3 , and 4 MeO PCE ( 2b , 3b and 4b) 24 did not display this phenomenon, whereas the three N propyl substituted PCPr 25 counterparts ( 2c , 3c and 4c) formed this ion. Similarly, the most abundant m/z 189 26 species was observed in the spectrum of 4 MeO PCPr (4c ) (Figure 4L1), whereas it 27 28 showed a relatively low abundance under quadrupole conditions (supplementary + 29 information). The CI IT MS spectra (Figures 3 and 4) yielded the expected [M + H] 30 ions and additional formation of an even electron ion following the loss of the 31 corresponding amine. In this case, the associated ions were formed at m/z 165 (1a) – 32 (1c), m/z 189 (2a) – (4c) and m/z 173 (5a) – (5c), respectively (Figure 5C) and were 33 presumably similar to the species observed under electrospray ionization conditions 34 35 (see below). 36 37 Interestingly, the CI IT mass spectra recorded for 2 methoxyphenyl substituted 38 substances (2a , 3a and 4a) differed from their isomeric representatives by the 39 relative abundance of the m/z 189 ion and the corresponding protonated molecules. 40 The meta and para substituted compounds appeared to display a decrease in 41 + 42 relative abundance of [M + H] relative to m/z 189 in the order of 2 MeO PCMe ( 2a ) 43 (Figure 3D2) > 3 MeO PCMe ( 3a ) (Figure 3G2) > 4 MeO PCMe ( 4a ) (Figure 4J2), 44 respectively. A potential reason for what appeared to be increased stability of 2 MeO 45 PCMe ( 2a ) may have been related to stabilization and increased charge distribution, 46 thus, possibly rendering the protonated molecule more stable (Figure 5D). In 47 48 addition, all CI IT mass spectra obtained from the three 2 methoxyphenyl analogs 49 also displayed an ion at m/z 121 that was either less prominent or absent in the 50 spectra of the meta and para substituted analogs. Figure 5E depicts an attempt to 51 rationalize its formation, which might require the presence of the 2 methoxyphenyl 52 position. 53
54 55 Representative electrospray ionization (ESI) triple quadrupole (QqQ) tandem mass 56 spectra and ion ratios for the 2 MeO , 3 MeO , and 4 MeO PCE isomers ( 2b , 3b and 57 4b) following direct infusion are presented in Figure 6A−C. Collision induced 58 dissociation (CID) of the product ions gave primarily rise of two product ions at m/z 59 60 http://mc.manuscriptcentral.com/dta 12 Drug Testing and Analysis Page 14 of 34
1 2 3 189 and m/z 121 that might be suitable for implementing the corresponding ion 4 transitions for screening purposes. In case of 3 MeO PCE ( 3b ), these two main ions 5 were also observed previously using a triple quadrupole linear ion trap mass 6 spectrometer. Correspondingly, the remaining ring substituted PCMe ( 2a , 3a and 4a) 7 8 and PCPr isomers ( 2c , 3c and 4c) also yielded the m/z 189 and m/z 121 ions 9 (supplemental data). Examination of the QqQ spectra in Figure 6A−C also revealed 10 the presence of a product ion at m/z 81 in the spectra of 3 MeO , and 4 MeO PCE 11 (3b , 4b ) that was absent in the QqQ tandem mass spectrum of 2 MeO PCE (2b ) 12 (Figure 6A). Given that the tandem mass spectra were recorded under identical 13 14 conditions and collision energies, it was tempting to consider the potential for 15 differentiation between the three PCE isomers based on distinct ion ratios alone. The 16 QqQ tandem mass spectrum of 3 MeO PCE (3b ) showed an increased abundance 17 of the m/z 81 species compared to 4 MeO PCE (4b ) (Figure 6B/6C). Interestingly, 18 the differential Forformation of Peerm/z 81 was equally Review observed in the tandem mass spectra 19 of the ring substituted PCMe ( 2a , 3a and 4a) and PCPr isomers ( 2c , 3c and 4c) as 20 21 well (supplemental data). The ultra high performance liquid chromatography 22 (UHPLC) ESI quadrupole time of flight (Q TOF) tandem mass spectra for 3 MeO , 23 and 4 MeO PCE (2b , 3b) are shown in Figure 6D/7E. Due to extensive in source CID 24 of the protonated molecule of 4 MeO PCE ( 3b ) (Figure 6F), a tandem mass 25 spectrum of the m/z 189.12746 base peak was recorded as shown in Figure 6G. As 26 observed in the QqQ tandem mass spectra of the 12 remaining N alkyl 27 28 arylcyclohexylamines (supplementary information), formation of the suggested 29 cyclohexen 1 ylium ion at m/z 81 was also frequently detected. The acquisition of 30 high mass accuracy Q TOF data was considered helpful for the proposal of 31 suggested structural representations associated with m/z 81, m/z 121 and m/z 189, 32 respectively (Figure 7). 33 34 35 Implementation of the HPLC DAD and UHPLC Q TOF MS methods provided 36 separation of the 2 MeO substituted N alkyl arylcyclohexylamines (2a −2c ) from their 37 3 MeO , and 4 MeO counterparts (3a −3c ) and ( 4a −4c ), respectively. Co elution, 38 however, was observed for the latter two groups of meta and para substituted 39 40 methoxyphenyl candidates (Figure 6 and supplementary information). Figure 8 41 shows the retention times and overlaid diode array spectra (DAD) obtained from PCE 42 isomers ( 2b ), ( 3b ) and ( 4b ), which demonstrated that the availability of full scan 43 ultraviolet information facilitated differentiation between the co eluting isomers. 44 Similarly, the differentiation between the remaining 3 methoxy and 4 methoxyphenyl 45 isomers ( 3a /4a ) and ( 3c /4c ) was also successful under DAD conditions and the 46 47 corresponding spectra are shown as supplementary information. The use of HPLC 48 DAD has been increasingly helpful when applied to a number of investigations that 49 investigated the presence and characterization of ring substituted isomers. [6,35] In 50 addition, the infrared spectra for all substances have been supplied as 51 supplementary information, which confirmed that the isomeric 2 , 3 and 4 methoxy 52 phenylcyclohexylamines could be differentiated, for example in the form of shifting 53 [36] 54 wavenumbers associated 1,2 , 1,3 , and 1,4 disubstituted benzenes. This was in 55 agreement with shift changes observed with 2 , 3 , and 4 methylphenyl substituted 56 PCP isomers. [37] 57 58 59 60 http://mc.manuscriptcentral.com/dta 13 Page 15 of 34 Drug Testing and Analysis
1 2 3 Nuclear magnetic resonance spectroscopy 4 5 Chemical shifts were assigned using chemical shift position, splitting pattern, 13 C 6 polarization enhancement nurtured during attached nucleus testing (PENDANT) and 7 8 2 D techniques (HMQC, HMBC, COSY 90) similar to the approach taken previously [4] 9 for tertiary amine based PCP and PCPy derivatives. A representative example of 10 an HMQC spectrum for 3 MeO PCPr ( 3c ) is shown in Figure 9. While some of the 11 present compounds have been described for a range of investigations, limited 12 information appeared available on the analytical properties including detailed NMR 13 analysis. For example, NMR data were reported for thiophene compounds ( 1a ) – 14 [33,38] [20] 15 (1c ) and 3 MeO PCE ( 3b ) but without chemical shift assignments, which are 16 provided here. The NMR data associated with the primary amine intermediates and a 17 more detailed discussion of the NMR chemical shift behavior is presented in the 18 supplemental section.For Peer Review 19 20 21 Proton chemical shifts 22 23 All proton chemical shift values, multiplicities and assignments for 24 arylcyclohexylamines ( 1a ) – (5c ) are summarized in Tables 1–3. 25 26 Identification of positional isomers is an important requirement of forensic 27 28 identification. The three positional methoxyphenyl isomers could be readily 1 29 distinguished by inspecting the downfield aromatic regions of the H proton spectra. 30 Aromatic multiplicities exhibited 2J and 3 4J couplings. The multiplets linked to the 31 para substituted isomers were readily identified by a characteristic pair of doublets of 32 multiplets that represented the aromatic protons due to additional splitting of the 33 doublets by long range couplings. The chemical shifts obtained from 2 , and 3 34 35 methoxyphenyl isomers were readily distinguishable in all equivalent N alkyl pairs as 36 a reflection of the position on the phenyl ring (Tables 1–3). 37 38 Carbon chemical shifts 39 40 41 All chemical shift data are summarized in Tables 4–6. 42 43 13 C chemical shifts were also useful in distinguishing the methoxyphenyl isomers. 44 Due to the symmetry of para substituted aryl rings, the 4 methoxyphenyl isomers 45 13 displayed four separate aromatic C chemical shifts; C 1’ , C 2’ C6’ , C 3’ C5’ , and C 4’ . The 46 2 and 3 methoxyphenyl counterparts showed six distinct aromatic 13 C chemical 47 48 shifts. Another distinguishing feature was the C 1’ chemical shift value. In the spectra 49 of 2 and 4 methoxyphenyl isomers, an upfield shift >10 ppm was observed when 50 compared to the meta substituted series, presumably due to enhanced shielding and 51 electron density from resonance contributions. 52 53 54 Conclusion 55 56 Newly emerging psychoactive substances continue to remain a challenge for forensic 57 scientists, clinicians and policy makers. The syntheses and comprehensive analytical 58 59 60 http://mc.manuscriptcentral.com/dta 14 Drug Testing and Analysis Page 16 of 34
1 2 3 characterizations of 15 N alkyl arylcyclohexylamines included a range of ‘research 4 chemical’ analogues that have not yet been described in detail. It was also 5 demonstrated that positional isomers could be differentiated using analytical 6 techniques typically involved in forensic and clinical casework. 7 8 9 References 10 11 12 [1] H. Morris, J. Wallach. From PCP to MXE: a comprehensive review of the non 13 medical use of dissociative drugs. Drug Test. Anal. 2014 , 6, 614. 14 15 16 [2] G. McLaughlin, N. Morris, P.V. Kavanagh, J.D. Power, J. O'Brien, B. Talbot, 17 S.P. Elliott, J. Wallach, K. Hoang, H. Morris, S.D. Brandt. Test purchase, synthesis 18 and characterizationFor of 2 methoxydiphenidine Peer Review (MXP) and differentiation from its 19 meta and para substituted isomers. Drug Test. Anal. 2015 , in press: doi: 20 10.1002/dta.1800. 21 22 [3] J. Wallach, P.V. Kavanagh, G. McLaughlin, N. Morris, J.D. Power, S.P. Elliott, 23 M.S. Mercier, D. Lodge, H. Morris, N.M. Dempster, S.D. Brandt. Preparation and 24 characterization of the 'research chemical' diphenidine, its pyrrolidine analogue, and 25 their 2,2 diphenylethyl isomers. Drug Test. Anal. 2015 , 7, 358. 26 27 28 [4] J. Wallach, G. De Paoli, A. Adejare, S.D. Brandt. Preparation and analytical 29 characterization of 1 (1 phenylcyclohexyl)piperidine (PCP) and 1 (1 30 phenylcyclohexyl)pyrrolidine (PCPy) analogues. Drug Test. Anal. 2014 , 6, 633. 31 32 [5] The Misuse of Drugs Act 1971 (Amendment) Order 2013 No. 239. Available 33 at: [06 June 34 http://www.legislation.gov.uk/uksi/2013/239/pdfs/uksi_20130239_en.pdf 35 2015]. 36 37 [6] S.P. Elliott, S.D. Brandt, J. Wallach, H. Morris, P.V. Kavanagh. First reported 38 fatalities associated with the ‘research chemical’ 2 methoxydiphenidine. J. Anal. 39 Toxicol. 2015 , 39 , 287. 40 41 42 [7] A. Helander, O. Beck, M. Bäckberg. Intoxications by the dissociative new 43 psychoactive substances diphenidine and methoxphenidine. Clin. Toxicol. 2015 , 53 , 44 446. 45 46 [8] K.E. Hofer, C. Degrandi, D.M. Müller, U. Zürrer Härdi, S. Wahl, C. Rauber 47 Lüthy, A. Ceschi. Acute toxicity associated with the recreational use of the novel 48 dissociative psychoactive substance methoxphenidine. Clin. Toxicol. 2014 , 52 , 1288. 49 50 51 [9] European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). New 52 psychoactive substances in Europe. An update from the EU Early Warning System 53 (March 2015). Lisbon, 2015. Available at: 54 http://www.emcdda.europa.eu/attachements.cfm/att_235958_EN_TD0415135ENN.p 55 df [06 June 2015] 56 57 58 59 60 http://mc.manuscriptcentral.com/dta 15 Page 17 of 34 Drug Testing and Analysis
1 2 3 [10] S.D. Brandt, L.A. King, M. Evans Brown. The new drug phenomenon. Drug 4 Test. Anal. 2014 , 6, 587. 5 6 [11] Z. Ates Alagoz, A. Adejare. NMDA receptor antagonists for treatment of 7 depression. Pharmaceuticals 2013 , 6, 480. 8 9 10 [12] L.V. Kalia, S.K. Kalia, M.W. Salter. NMDA receptors in clinical neurology: 11 excitatory times ahead. Lancet Neurol. 2008 , 7, 742. 12 13 [13] S.A. Lipton. Paradigm shift in neuroprotection by NMDA receptor blockade: 14 memantine and beyond. Nat. Rev. Drug Discov. 2006 , 5, 160. 15 16 17 [14] K.W. Muir. Glutamate based therapeutic approaches: clinical trials with 18 NMDA antagonists.For Curr. Opin.Peer Pharmacol. Review 2006 , 6, 53. 19 20 [15] B.L. Roth, S. Gibbons, W. Arunotayanun, X.P. Huang, V. Setola, R. Treble, L. 21 Iversen. The ketamine analogue methoxetamine and 3 and 4 methoxy analogues of 22 phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor. 23 PloS One 2013 , 8, e59334. 24 25 26 [16] C. Sauer, F.T. Peters, R.F. Staack, G. Fritschi, H.H. Maurer. New designer 27 drugs N (1 phenylcyclohexyl) 2 ethoxyethanamine (PCEEA) and N (1 28 phenylcyclohexyl) 2 methoxyethanamine (PCMEA): Studies on their metabolism and 29 toxicological detection in rat urine using gas chromatographic/mass spectrometric 30 techniques. J. Mass Spectrom. 2008 , 43 , 305. 31 32 [17] P. Rösner, T. Junge, G. Fritschi, B. Klein. Neue synthetische Drogen: 33 34 Piperazin , Propicyclidin und α Aminopropiophenonderivate. Toxichem Krimtech 35 1999 , 66 , 81. 36 37 [18] C. Sauer, F.T. Peters, R.F. Staack, G. Fritschi, H.H. Maurer. New designer 38 drug N (1 phenylcyclohexyl) 3 ethoxypropanamine (PCEPA): Studies on its 39 metabolism and toxicological detection in rat urine using gas chromatographic/mass 40 spectrometric techniques. J. Mass Spectrom. 2006 , 41 , 1014. 41 42 43 [19] European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). 44 EMCDDA–Europol 2010 Annual Report on the implementation of Council Decision 45 2005/387/JHA. In accordance with Article 10 of Council Decision 2005/387/JHA on 46 the information exchange, risk assessment and control of new psychoactive 47 substances. Lisbon, 2011 . Available at: 48 http://www.emcdda.europa.eu/attachements.cfm/att_132857_EN_EMCDDA-Europol 49 Annual Report 2010A.pdf [06 June 2015]. 50 51 [20] G. De Paoli, S.D. Brandt, J. Wallach, R.P. Archer, D.J. Pounder. From the 52 street to the laboratory: analytical profiles of methoxetamine, 3 methoxyeticyclidine 53 and 3 methoxyphencyclidine and their determination in three biological matrices. J. 54 Anal. Toxicol. 2013 , 37 , 277. 55 56 57 [21] European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). New 58 drugs in Europe, 2012. EMCDDA Europol 2012 Annual Report on the 59 60 http://mc.manuscriptcentral.com/dta 16 Drug Testing and Analysis Page 18 of 34
1 2 3 implementation of Council Decision 2005/387/JHA. Lisbon. 2013 . Available at: 4 http://www.emcdda.europa.eu/attachements.cfm/att_212366_EN_EMCDDA-Europol 5 2012 Annual Report_final.pdf [06 June 2015]. 6 7 8 [22] R. F. Parcell. Heterocyclic compounds and methods for producing the same. 9 Patent US2921076A. Parke, Davis & Co. 1960 . 10 11 [23] A. Thurkauf, B. de Costa, S. Yamaguchi, M.V. Mattson, A.E. Jacobson, K.C. 12 Rice, M.A. Rogawski. Synthesis and anticonvulsant activity of 1 13 phenylcyclohexylamine analogs. J. Med. Chem. 1990 , 33 , 1452. 14 15 16 [24] V.H. Maddox, E.F. Godefroi, R.F. Parcell. The synthesis of phencyclidine and 17 other 1 arylcyclohexylamines. J. Med. Chem. 1965 , 8, 230. 18 For Peer Review 19 [25] S.P. Elliott, S.D. Brandt, S. Freeman, R.P. Archer. AMT (3 (2 20 aminopropyl)indole) and 5 IT (5 (2 aminopropyl)indole): an analytical challenge and 21 implications for forensic analysis. Drug Test. Anal. 2013 , 5, 196. 22 23 24 [26] Y.N.A. Soh, S. Elliott. An investigation of the stability of emerging new 25 psychoactive substances. Drug Test. Anal. 2013 , 6, 696. 26 27 [27] A.C. Allen, J. Robles, W. Dovenski, S. Calderon. PCP: a review of synthetic 28 methods for forensic clandestine investigation. Forensic Sci. Int. 1993 , 61 , 85. 29 30 31 [28] P. Geneste, P. Herrmann, J.M. Kamenka, A. Pons. Nouvelles voies d'accès 32 aux isomères des phényl 1 cyclohexylamines substituées au cyclohexane. Bull. Soc. 33 Chim. France 1975 , 7, 1619. 34 35 [29] M. Rogawski, A. Thurkauf, K. Rice, A. Jacobsen, J. French Mullen, 36 Anticonvulsant activity of phencyclidine analogs: structural modifications resulting in 37 enhanced seizure protection relative to motor side effects. In Frontiers in excitatory 38 amino acid research: proceedings of an international symposium "Excitatory Acids 39 '88," Manaus, Amazonas, Brazil, March 28-April 2, 1988 , (Ed.: L. Turski, J. Lehmann, 40 E.A. Cavalheiro), Liss, New York, 1988 , pp. 227. 41 42 43 [30] S.O. Casalotti, A.P. Kozikowski, A. Fauq, W. Tückmantel, K.E. Krueger. 44 Design of an irreversible affinity ligand for the phencyclidine recognition site on N 45 methyl D aspartate type glutamate receptors. J. Pharmacol. Exp. Ther. 1992 , 260 , 46 21. 47 48 [31] V. John, R. Hom, J. Sealy, J. Aquino, G. Probst, J. Tung, L. Fang. Methods of 49 treatment of amyloidosis using aspartyl protease inihibitors. Patent. 50 WO2005070407A1, Elan Pharmaceuticals, Inc. 2005 . 51 52 53 [32] J.K. Reel, J.D. Leander, L.G. Mendelsohn, D.D. Schoepp, P.L. Ornstein, D.A. 54 Evrard, R.B. Hermann, D.M. Zimmerman, The search for a PCP antagonist: 55 synthesis and characterization of novel arylcyclohexylamine derivatives. In Sigma 56 and Phencyclidine-Like Compounds as Molecular Probes in Biology , (Ed.: E.F. 57 Domino, J.M. Kamenka), Npp Books, Ann Arbor, MI, 1988 , pp. 27. 58 59 60 http://mc.manuscriptcentral.com/dta 17 Page 19 of 34 Drug Testing and Analysis
1 2 3 [33] X. Ouyang, J. Mukherjee, Z. Y. Yang. Synthesis, radiosynthesis, and 4 biological evaluation of fluorinated thienylcyclohexyl piperidine derivatives as 5 potential radiotracers for the NMDA receptor linked calcium ionophore. Nucl. Med. 6 Biol. 1996 , 23 , 315. 7 8 [34] A.D. Westwell, A. Hutchings, D.G.E. Caldicott. The identification and chemical 9 characterization of a new arylcyclohexylamine, methoxetamine, using a novel 10 Emergency Department toxicosurveillance tool. Drug Test. Anal. 2013 , 5, 203. 11 12 13 [35] S.D. Brandt, S.P. Elliott, P.V. Kavanagh, N.M. Dempster, M.R. Meyer, H.H. 14 Maurer, D.E. Nichols. Analytical characterization of bioactive N benzyl substituted 15 phenethylamines and 5 methoxytryptamines. Rapid Commun. Mass Spectrom. 2015 , 16 29 , 573. 17 18 For Peer Review 19 [36] D.H. Williams, I. Fleming, Spectroscopic Methods in Organic Chemistry, Fifth 20 Edition , McGraw Hill, Berkshire, UK, 1995. 21 22 [37] B.A. Lodge, R. Duhaime, J. Zamecnik, P. Macmurray, R. Brousseau. New 23 street analogs of phencyclidine. Forensic Sci. Int. 1992 , 55 , 13. 24 25 26 [38] J. M. Kamenka, J. Hamon, J. Vignon. Phencyclidine derivatives, preparation 27 method and pharmaceutical compositions containing same. Patent. US6342511B1. 28 Société de Conseils de Recherches et d'Applications Scientifiques. 2002 . 29 30 31 Figure captions 32 33 Figure 1. Phencyclidine (PCP) and other representative arylcyclohexylamine and 1,2 34 diphenylethylamine examples associated with the recreational ‘research chemicals’ 35 36 market. 37 38 Figure 2. A: Fifteen N alkyl arylcyclohexylamines subjected to synthesis and 39 analytical characterizations. B: Synthetic routes employed for the preparation of (1a ) 40 – (5c ) 41
42 43 Figure 3. Gas chromatography retention times and ion trap (IT) mass spectra 44 obtained in electron (EI) and chemical ionization (CI) mode. 45 46 Figure 4. Gas chromatography retention times and ion trap (IT) mass spectra 47 obtained in electron (EI) and chemical ionization (CI) mode. 48 49 50 Figure 5. Suggested key ions following mass spectral detection under EI and CI 51 conditions. 52 53 Figure 6. A C: Electrospray ionization triple quadrupole mass spectra of three 54 positional N ethyl 1 phenylcyclohexan 1 amine (PCE) isomers. D G. Ultra high 55 56 performance liquid chromatography quadrupole time of flight tandem mass spectra. 57 58 59 60 http://mc.manuscriptcentral.com/dta 18 Drug Testing and Analysis Page 20 of 34
1 2 3 Figure 7. Suggested key ions following mass spectral detection under electrospray 4 ionization conditions. 5 6 Figure 8. Ultraviolet full scan spectra of N ethyl 1 phenylcyclohexan 1 amine (PCE) 7 8 isomers by high performance liquid chromatography coupled to diode array detection. 9 10 Figure 9. Heteronuclear single quantum coherence spectrum (HSQC) of 3 MeO 11 PCPr. 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta 19 Page 21 of 34 Drug Testing and Analysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 Figure 1. Phencyclidine (PCP) and other representative arylcyclohexylamine and 1,2-diphenylethylamine 28 examples associated with the recreational ‘research chemicals’ market. 118x69mm (300 x 300 DPI) 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 22 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Figure 2. A: Fifteen N-alkyl-arylcyclohexylamines subjected to synthesis and analytical characterizations. B: 44 Synthetic routes employed for the preparation of (1a) – (5c) 45 207x225mm (300 x 300 DPI) 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Page 23 of 34 Drug Testing and Analysis 152 165 100% EI-IT-MS 100% CI-IT-MS 4.61 min HN (1a ) 75% S 75% 1 A1 A2 2 162 50% 165 (1a ) 50% 196 3 138 180 123 25% 195 25% 180 4 97 112 50% 0% 6 100 200 300 400 m/z 100 200 300 400 m/z 7 100% EI-IT-MS 166 100% CI-IT-MS 165 8 4.77 min (1b ) 75%9 165 75% B1 HN 10 B2 50% 176 S 50% 11 123 180 210 25% 209 (1b ) 25% 12 97 152 126 130% 0% 14 100 200 300 400 m/z 100 200 300 400 m/z 15 100% EI-IT-MS 180 100% CI-IT-MS 165 16 5.25 min (1c ) 75%17 75% C1 HN C2 18 For Peer Review224 50%19 165 S 50% 190 25%20 60 123 25% 58 97 223 (1c ) 58 140 21 152 0% 0% 22 23 100 200 300 400 m/z 100 200 300 400 m/z 176 189 100%24 EI-IT-MS 220 HN 100% CI-IT-MS 5.62 min (2a ) 220 75%25 75% 26 D1 D2 50%27 218 O 50% 148 204 121 28 121 189 25% 91 133 (2a ) 25% 29 77 152 300% 0% 31 100 200 300 400 m/z 100 200 300 400 m/z 234 234 100%32 EI-IT-MS 100% CI-IT-MS 189 33 5.72 min HN (2b ) 75% 190 75% 34 E1 E2 50%35 50% 36 232 O 25% 162 218 25% 121 121 37 91 147 204 (2b ) 380% 0% 39 100 200 300 400 m/z 100 200 300 400 m/z 40 248 248 100% EI-IT-MS 100% CI-IT-MS 41 6.15 min (2c ) 75%42 204 HN 75% F1 F2 189 43 50% 44 50% 246 O 60 147189 232 25%45 60 121 25% 58 121 58 176 46 91 134 218 (2c ) 0% 47 0% 100 200 300 400 m/z 100 200 300 400 m/z 48 EI-IT-MS 176 CI-IT-MS 189 100%49 100% 6.00 min (3a ) 50 HN 75% G1 75% G2 51 220 50%52 219 50% (3a ) 53 121 189 O 25% 112 162 204 25% 54 91 550% 0% 56 100 200 300 400 m/z 100 200 300 400 m/z 100%57 EI-IT-MS 190 100% CI-IT-MS 189 58 6.09 min (3b ) 75% HN 75% 59 H1 234 H2 234 50%60 233 50% 232 (3b ) 25% 126 176 http://mc.manuscriptcentral.com/dta25% 46 121 O 91 147 204 121 0% 0% 100 200 300 400 m/z 100 200 300 400 m/z Drug Testing and Analysis Page 24 of 34
1 2 3 4 5 6 7 EI-IT-MS 204 CI-IT-MS(3c ) 248 100%8 100% 189 9 6.49 min 60 I2 75%10 I1 HN 75% 248 50%11 247 50% 147 12 246 (3c ) 247 25%13 60 121 140 189 25% 58 58 91 134 218 O 140% 0% 15 100 200 300 400 m/z 100 200 300 400 m/z 189 100%16 EI-IT-MS 176 100% CI-IT-MS 4a 17 6.17 min HN ( ) 75% 75% 18 J1 189 For PeerJ2 Review 50%19 50% 20 162 219 (4a ) O 147 218 25%21 121 204 25% 91 112 218 220% 0% 23 100 200 300 400 m/z 100 200 300 400 m/z 100%24 EI-IT-MS 190 100% CI-IT-MS 189 25 6.26 min (4b ) 75% HN 75% 26 K1 K2 50%27 147 50% 28 233 134 232 (4b ) O 191 25% 121 176 25% 29 91 204 126 234 300% 0% 31 100 200 300 400 m/z 100 200 300 400 m/z 32 204 189 100% EI-IT-MS 189 100% CI-IT-MS 33 6.68 min (4c ) 75%34 HN 75% L1 L2 35 50% 50% 36 246 4c 248 25%37 121 147 ( ) O 25% 191 247 247 58 38 91 176 218 140 246 0% 0% 39 100 200 300 400 m/z 100 200 300 400 m/z 40 EI-IT-MS 160 CI-IT-MS 173 100% 100% 41 5.17 min (5a ) 42 HN 75% M1 75% M2 43 204 50%44 203 50% 146 202 (5a ) 204 45 131 188 203 25% 105 173 25% 202 46 91 105 470% 0% 48 100 200 300 400 m/z 100 200 300 400 m/z 100%49 EI-IT-MS 174 100% CI-IT-MS 173 5.25 min (5b ) 75%50 HN 75% N1 N2 51 218 50%52 217 50% 218 160 216 217 53 105 (5b ) 25% 131 188 25% 54 46 91 550% 0% 56 100 200 300 400 m/z 100 200 300 400 m/z 5c 100%57 EI-IT-MS 188 CI-IT-MS ( ) 173 100% 60 58 5.68 min O2 75%59 O1 HN 75% 232 232 231 50%60 231 50% 230 230 25% 60 105 173 (5c ) http://mc.manuscriptcentral.com/dta58 131 25% 58 91 216 188 0% 0% 100 200 300 400 m/z 100 200 300 400 m/z A (EI)Page 25 of 34 H Drug Testing and Analysis NH NH NH NH 2 1 R2 R2 2 R R 1 2 R1 R1 R1 R 3 4 1 2 R = 2-, 3-, 4-OCH 3 ; R = CH 3 : m/z 176 ( 2a , 3a , 4a ) 5 1 2 m/z 2b 3b 4b 6 R = 2-, 3-, 4-OCH 3 ; R = C 2 H 5 : 190 ( , , ) 1 2 m/z 2c 3c 4c 7 R = 2-, 3-, 4-OCH 3 ; R = C 3 H 7 : 204 ( , , ) 8 NH NH 9 S R 10 R 11 12 13 m/z 1a R = CH 3 : 152 ( ) R = CH : m/z 160 ( 5a ) 14 3 R = C 2 H 5 : m/z 166 ( 1b ) R = C H : m/z 174 ( 5b ) 15 2 5 R = C H : m/z 180 ( 1c ) R = C H : m/z 188 ( 5c ) 16 3 7 3 7 17 18 For Peer Review 19 B20 (EI)21 22 NH 23 NH 24 O - 25 CH 2O 26 m/z 27 H 189 28 29 4-MeO-PCMe (4a ) 30 m/z 219 31 32 C33 (CI)34 35 36 37 S 38 O 39 40 m/z 189 m/z 165 m/z 173 41 42 2-, 3-, 4-MeO-PCMe (2a , 3a, 4a) TCMe (1a ) 3-Me-PCMe (5a ) 43 2-, 3-, 4-MeO-PCE (2b , 3b, 4b) TCE (1b ) 3-Me-PCE (5b ) 44 2-, 3-, 4-MeO-PCPr (2c , 3c, 4c) TCPr (1c ) 3-Me-PCPr (5c ) 45 D46 (CI)47 48 49 H R 50 N m/z 189 51 H 52 O 53 54 55 E56 (CI)57 58 59 60 O NH 2 NH 2 http://mc.manuscriptcentral.com/dta R O O H m/z 121 A HN MS/MS m/z %BPI Drug Testing and Analysis 80.84 0.90Page 26 of 34 90.80 0.71 O % 189.06 91.04 0.69 1 92.77 0.60 2-MeO-PCE 93.01 0.56 2 2b 120.87 100.00 3 ( ) 0 189.06 38.71 4 40 60 80 100 120 140 160 180 200 220 240 260 m/z 5 H+ 6 189.04 m/z 100 %BPI 7 B HN 66.87 0.54 8 MS/MS 120.85 80.82 27.55 9 108.84 0.74 120.85 70.69 10
% 189.04 100.00 O 11 80.82 12 3-MeO-PCE 13 (3b ) 14 0 m/z %BPI 15 40 60 80 100 120 140 160 180 200 220 240 260 m/z 66.95 0.10 16 189.04 80.82 6.02 100 H+ 90.69 0.13 17 C 90.85 0.18 18 MS/MS 120.85For Peer ReviewHN 108.66 0.23 19 108.87 0.22 20 109.17 0.18 % 119.80 0.09 21 O 120.85 69.24 22 4-MeO-PCE 146.79 0.62 23 80.82 (4b ) 147.08 0.60 24 0 147.28 0.43 189.04 100.00 25 40 60 80 100 120 140 160 180 200 220 240 260 m/z 26 27+ESI Product Ion (2.303 min) Frag=110.0V [email protected] (234.18517[z=1] -> **) PCP 2b-MSMS.d x 10284 D 29 121.06449 1.530 MS/MS 131 32 2-MeO-PCE 0.5 91.05340 (2b ) 33 65.03877 77.03703 112.80389 148.08396 189.12725 034 m/z 35 55 65 75 85 95 105 115 125 135 145 155 165 175 185 195 205 215 225 235 36 37+ESI Product Ion (2.139 min) Frag=110.0V [email protected] (234.18520[z=1] -> **) PCP 3b-MSMS.d 38 x 10 4 39 121.06448 2.5 E 40 MS/MS 2 1.541 142 3-MeO-PCE 81.06968 189.12709 0.543 (3b ) 52.02949 65.03867 91.05415 109.06451 135.08162 147.07849 175.06716 044 45 55 65 75 85 95 105 115 125 135 145 155 165 175 185 195 205 215 225 m/z 46 47+ESI Scan (2.160 min) Frag=130.0V PCP 4b-MS.d 5 (in-source CID) x 1048 849 F 189.12728 MS 650 4-MeO-PCE 451 121.06432 (4b ) 52 129.10176 2 224.12803 53 102.12766 102.12766 139.04975 147.07980 158.96089 178.12243 215.17529 239.22485 054 105 115 125 135 145 155 165 175 185 195 205 215 225 235 m/z 55 56+ESI Product Ion (2.163 min) Frag=110.0V [email protected] (189.12746[z=1] -> **) PCP 4b-MSMS.d 574 x 10 858 G 121.06421 59 6 MS/MS 60 4 4-MeO-PCE http://mc.manuscriptcentral.com/dta (4b ) 2 189.12727 55.01694 65.27901 81.06908 91.05323 115.05398 135.08020 147.08080 160.08871 177.91109 0 55 65 75 85 95 105 115 125 135 145 155 165 175 185 195 m/z Page 27 of 34 Drug Testing and Analysis
1 2 3 4 5 6 7 8 9 10 11 12 13 R 14 N 15 H2 H 16 O O + 17 C6H9 18 m/z 81.06988 (calc.) For Peer +Review 19 C13 H17 O m/z 20 R = CH 3 : 220 m/z 189.12739 (calc.) 21 R = C 2 H 5 : m/z 234 22 R = C H : m/z 248 23 3 7 24 2a 3a 4a 2-,25 3-, 4-MeO-PCMe ( , , ) 2b 3b 4b 2-,26 3-, 4-MeO-PCE ( , , ) 2-,27 3-, 4-MeO-PCPr (2c , 3c, 4c) 28 29 30 O O 31 32 33 34 35 36 37 38 39 O 40 O 41 42 43 44 45 46 O O 47 48 49 50 51 52 − 53 54 H H (C H ) 55 R 5 8 56 N H 57 2 O O O 58 O 59 60 + C8H9O http://mc.manuscriptcentral.com/dta m/z 121.06479 (calc.) + C13 H17 O m/z 189.12739 (calc.) Drug Testing and Analysis Page 28 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 % 46 HN HN 10047 HN 48 49 8050 O O 51 226.9 O 52 6053 7.68 min 7.45 min 7.42 min 54 217.8 2-MeO-PCE 3-MeO-PCE 4-MeO-PCE 55 (2b ) (3b ) (4b ) 4056 217.1 57 58 272.6 2059 275.7 60 271.3 http://mc.manuscriptcentral.com/dta 0 nm 200 250 300 350 400 450 500 550 594 Page 29 of 34 Drug Testing and Analysis
1 2 3 4 H5’ H2’ H 5 6’ H4’ 6 7 8 9 105 10 110 11 C2’ 12 C4’ 115 13 14C6’ 120 15 125 16 γ 17 C5’ 130 18 For Peer Reviewβ 19 α 135 ppm 20 C1’ 21 6 NH 140 2’ 22 5 1 C 23 1’ c 145 24 3’ 4 2 150 25 6’ 3 4’ 26 155 27 5’ 28 160 C3’ 29 30 7.70 7.60 7.50 7.40 7.30 7.20 7.10 7.00 6.90 6.80 6.70 ppm 31 32 33 Cc 34 ()OCH3 H H4 γ 35 H4 36 (H ax ) H (H eq ) 3,5 37 (H ax ) 38 H3,5 39 H2,6 H2,6 (H eq ) (H ) (H ) Hβ 40 eq Hα ax 41 42 43 44 45 10 Cγ 46 47 48 Cβ 20 C3,5 49 C 50 4 51 30 52 C2,6 53 ppm 54 40 55 Cα 56 57 50 58C 59c ()OCH60 3 60 C1 http://mc.manuscriptcentral.com/dta
4.00 3.60 3.20 2.80 2.40 2.00 1.60 1.20 0.80 ppm Drug Testing and Analysis Page 30 of 34
1 2 3 4 5
6 1 7 Table 1. 400 MHz H NMR spectra of HCl salts in CDCl 3. γ γ γ 8 β β β 9 α α α 10 NH NH NH 11 C6 C6 C 12 6 C5 C1 C2' C5 C1 C2' Cc C5 C1 C1' C3' C1' C3' S 13 C1' C4 C2 Cc C4 C2 For Peer ReviewC3 C6' C4' C3 C6' C4' C C 14 4 2 C4' C C5' C5' 15 3 C2' C3' 16Proton TCMe ( 1a ) 2 MeO PCMe ( 2a ) 3 MeO PCMe ( 3a ) 4 MeO PCMe ( 4a ) 3 Me PCMe ( 5a ) 17H 2.59 d (13.0 Hz, 2H ) 2.67 d (12.7 Hz, 2H ) 2.61 d (12.9 Hz, 2H ) 2.62 d (12.8 Hz, 2H ) 2.62 d (13.2 Hz, 2H ) 18 2,6 eq eq eq eq eq 2.32–2.18 m (2H ) 2.51–2.24 m (2H ) 2.41–2.12 m (2H ) 2.28–2.15 m (2H ) 2.33–2.19 m (2H ) 19 ax ax ax ax ax 20H3,5 1.92–1.73 m (2H eq ) 1.99–1.80 m (2H eq ) 2.03–1.76 m (2H eq ) 1.90–1.75 m (2 H eq ) 1.93–1.77 m (2H eq ) 21 1.60–1.34 m (2Hax ) 1.46–1.27 m (2H ax ) 1.45–1.31 m (2H ax ) 1.35 m (2H ax ) 1.38 qt (13.4, 3.4 Hz, 22 2H ax ) 23H4 1.66–1.54 m (1Heq ) 1.71–1.56 m (1H eq ) 1.64–1.45 m (1H eq ) 1.63–1.52 m (1H eq ) 1.64–1.45 m (2H) 24 1.60–1.34 m (1Hax ) 1.53 qt (11.7, 3.8 Hz, 1H ax ) 1.52 qt (12.6, 3.6 Hz, 1H ax ) 1.48 qt (12.8, 3.7 Hz, 1H ax )
25H1’ – – – – –
26H2’ 7.42 dd (3.7, 1.2 Hz) – 7.37 t (2.2 Hz) 7.64 dm (8.9 Hz) 7.57 s
27H3’ 7.09 dd (5.1, 3.7 Hz) 6.99 dd (8.0, 1.0 Hz) – 6.98 dm (8.9 Hz) – 28 H4’ 7.38 dd (5.1, 1.2 Hz) 7.42–7.35 m 6.91 ddd (8.3, 2.5, 1.0 Hz) – 7.50 d (7.8 Hz) 29H 7.03 td (7.6, 1.2 Hz) 7.34 t (8.0 Hz) 6.98 dm (8.9 Hz) 7.34 t (7.8 Hz) 30 5’ H – 7.42–7.35 m 7.22 ddd (7.8, 2.0, 0.9 Hz) 7.64 dm (8.9 Hz) 7.18 d (7.8 Hz) 31 6’ 32Hα 2.34 s (CH 3) 2.32 s (CH 3) 2.23 s (CH 3) 2.23 s (CH 3) 2.23 s (CH 3) 33Hβ – – – – – 34Hγ – – – – – 35Hc – 4.04 s (OCH 3) 3.90 s (OCH 3) 3.83 s (OCH 3) 2.62 s (CH 3) + + + + + + 36NH 9.73 s (NH 2 ) 9.30 s (NH 2 ) 9.60 s (NH 2 ) 9.53 s (NH 2 ) 9.58 s (NH 2 ) 37* b = broad; d = doublet; m = multiplet; s = singlet; triplet; q = quartet; quint = quintet; sex = sextet 38 39 40 41 42 43 44 45 46 http://mc.manuscriptcentral.com/dta 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 31 of 34 Drug Testing and Analysis
1 2 3 4 5
6 1 7 Table 2. 400 MHz H NMR spectra of HCl salts in CDCl 3. 8 Proton TCE ( 1b ) 2 MeO PCE ( 2b ) 3 MeO PCE ( 3b ) 4 MeO PCE ( 4b ) 3 Me PCE ( 5b ) 9 H2,6 2.73 d (13.0 Hz, 2H eq ) 2.61 d (12.7 Hz, 2H eq ) 2.77 d (13.0 Hz, 2H eq ) 2.80 d (12.6 Hz, 2H eq ) 2.81 d (12.6 Hz, 2H eq ) 10 2.39 td (12.5, 3.7 Hz, 2H ax ) 2.45 td (12.6, 3.9 Hz, 2H ax ) 2.34 td (13.3, 3.7 Hz, 2H ax ) 2.33 td (13.1, 3.4 Hz, 2H ax ) 2.35 td (13.1, 3.6 Hz, 11 2H ax )
12H3,5 1.86–1.72 m (2H eq ) 1.94–1.82 m (2H eq ) 1.88–1.72 m (2H eq ) 1.89–1.70 m (2H eq ) 1.87–1.72 m (2H eq ) 13 1.54–1.34 m (2H ax ) 1.46–1.28For m (2H ax ) Peer1.35 qt (12.8,Review 3.3 Hz, 2H ax ) 1.29 qt (12.9, 3.4 Hax ) 1.31 qt (13.0, 3.2 Hz, 14 2H ax ) 15 H4 1.69–1.53 m (1H eq ) 1.65–1.57 m (1H eq ) 1.67–1.56 m (1H eq ) 1.66–1.54 m (1H eq ) 1.70–1.54 m (1H eq ) 16 1.54–1.34 m (1H ) 1.52 qt (11.7, 4.1 Hz, 1 H ) 1.48 qt (12.4, 3.8 Hz, 1H ) 1.48 qt (12.8, 3.8 Hz, 1H ) 1.47 qt (12.4, 3.8 Hz, 17 ax ax ax ax 1H ) 18 ax H – – – – – 19 1’ 20H2’ 7.48 dd (3.8, 1.1 Hz) – 7.47 t (2.1 Hz) 7.71 dm (8.9 Hz) 7.65 s 21H3’ 7.10 dd (5.2, 3.6 Hz) 7.01 dd (8.0, 0.9 Hz) – 6.99 dm (8.9 Hz) – 22H4’ 7.39 dd (5.2, 1.1 Hz) 7.40–7.35 m 6.92 ddd (8.1, 2.5, 1.0 Hz) – 7.57 d (7.9 Hz) 23H5’ 7.06 td (7.6, 1.1 Hz) 7.36 t (8.0 Hz) 6.99 dm (8.9 Hz) 7.35 t (7.7 Hz)
24H6’ – 7.40–7.35 m 7.30 d (7.7 Hz) 7.71 dm (8.9 Hz) 7.18 d (7.6 Hz)
25Hα 2.80–2.68 m (CH 2) 2.69 bs (CH 2) 2.69–2.53 m (CH 2) 2.68 2.53 m (CH 2) 2.67–2.51 m (CH 2) 26 Hβ 1.42 t (7.3 Hz, CH 3) 1.36 t (7.3 Hz, CH 3) 1.38 t (7.3 Hz, CH 3) 1.29 t (7.3 Hz, CH 3) 1.38 t (7.3 Hz, CH 3) 27H – – – – – 28 γ Hc – 4.01 s (OCH 3) 3.91 s (OCH 3) 3.83 s (OCH 3) 2.43 s (CH 3) 29 + + + + + + 30NH 9.63 s (NH 2 ) 9.20 bs (NH 2 ) 9.53 s (NH 2 ) 9.45 s (NH 2 ) 9.50 s (NH 2 ) 31* b = broad; d = doublet; m = multiplet; s = singlet; triplet; q = quartet; quint = quintet; sex = sextet. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 http://mc.manuscriptcentral.com/dta 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Drug Testing and Analysis Page 32 of 34
1 2 3 4 5
6 1 7 Table 3. 400 MHz H NMR spectra of HCl salts in CDCl 3. 8 Proton TCPr ( 1c ) 2 MeO PCPr ( 2c ) 3 MeO PCPr ( 3c ) 4 MeO PCPr ( 4c ) 3 Me PCPr ( 5c ) 9 H2,6 2.73 d (12.7 Hz, 2H eq ) 2.65–2.50 m (2H eq ) 2.78 d (12.6 Hz, 2H eq ) 2.79 d (12.5 Hz, 2H eq ) 2.81 d (12.6 Hz, 2H eq ) 10 2.47–2.32 m (2H ax ) 2.45 td (13.0, 3.6 Hz, 2H ax ) 2.35 td (13.0, 3.5 Hz, 2H ax ) 2.34 td (12.9, 3.5 Hz, 2H ax ) 2.35 td (13.0, 3.5 Hz, 11 2H ax )
12H3,5 1.85–1.74 m (2H eq ) 1.93–1.76 m (2H eq ) 1.83–1.71 m (2H eq ) 1.81–1.69 m (2H eq ) 1.82–1.70 m (2H eq ) 13 1.53–1.37 m (2H ax ) 1.46–1.19For m (2Hax ) Peer1.34 qtReview (13.0, 3.5 Hz, 2H ax ) 1.29 qt (13.2, 3.4 Hz, 2H ax ) 1.29 qt (12.7, 3.5 Hz, 14 2H ax ) 15 16H 1.66–1.56 m (1H ) 1.66–1.57 m (1H ) 1.70–1.53 m (1H ) 1.65–1.53 m (1H ) 1.64–1.54 m (1H ) 17 4 eq eq eq eq eq 1.53–1.37 m (1H ) 1.51 qt (12.3, 3.7 Hz, 1H ) 1.46 qt (12.5, 3.7 Hz, 1H ) 1.47 qt (12.6, 3.8 Hz, 1H ) 1.48 qt (12.8, 3.7 Hz, 18 ax ax ax ax 1H ) 19 ax 20H1’ – – – – – 21H2’ 7.48 dd (3.6, 1.4 Hz) – 7.47 t (2.1 Hz) 7.71 dm (8.9 Hz) 7.66 s 22H3’ 7.10 dd (5.1, 3.5 Hz) 7.01 dd (8.8, 1.2 Hz) – 6.99 dm (8.9 Hz) – 23H4’ 7.39 dd (5.2, 1.2 Hz) 7.45–7.37 m 6.92 ddd (8.0, 2.3, 0.9 Hz) – 7.56 d (8.0 Hz)
24H5’ 7.06 td (7.6, 1.2 Hz) 7.36 t (8.0 Hz) 6.99 dm (8.9 Hz) 7.35 t (7.7 Hz)
25H6’ – 7.45–7.37 m 7.32–7.25 m 7.71 dm (8.9 Hz) 7.19 d (7.7 Hz) 26 Hα 2.66–2.51 m (CH 2) 2.65–2.50 m (CH 2) 2.50–2.40 m (CH 2) 2.50–2.39 m (CH 2) 2.46–2.40 m (CH 2) 27H 1.98–1.83 m (CH ) 1.93–1.76 m (CH ) 1.97–1.83 m (CH ) 1.96–1.81 m (CH ) 1.96–1.82 m (CH ) 28 β 2 2 2 2 2 H 0.84 t (7.5 Hz, CH ) 0.83 t (7.4 Hz, CH ) 0.80 t (7.4 Hz, CH ) 0.79 t (7.4 Hz, CH ) 0.79 t (7.7 Hz, CH ) 29 γ 3 3 3 3 3 Hc – 3.98 s (OCH 3) 3.91 s (OCH 3) 3.84 s (OCH 3) 2.43 s (CH 3) 30 + + + + + + 31NH 9.58 s (NH 2 ) 9.00 bs (NH 2 ) 9.47 s (NH 2 ) 9.38 s (NH 2 ) 9.44 s (NH 2 ) 32* b = broad; d = doublet; m = multiplet; s = singlet; triplet; q = quartet; quint = quintet; sex = sextet. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 http://mc.manuscriptcentral.com/dta 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 34 Drug Testing and Analysis
1 2 13 3 Table 4. 100 MHz C NMR spectra of HCl salts in CDCl 3. 4 Carbon TCMe ( 1a ) 2 MeO PCMe ( 2a ) 3 MeO PCMe ( 3a ) 4 MeO PCMe ( 4a ) 3 Me PCMe ( 5a ) 5 C1 61.95 64.08 63.64 63.26 63.49 6 C 34.68 32.42 33.28 33.21 33.16 7 2,6 8 C3,5 22.12 22.74 22.03 21.99 22.02 9 C4 24.66 25.06 24.96 25.90 25.01
10 C1’ 139.71 123.20 136.91 126.68 135.14 11 C 128.66 157.42 112.93 129.43 128.56 12 2’ 13 C3’ 127.81 111.59 160.48 114.44 139.02 14 C4’ 127.03 130.36 115.14 159.68 124.99 15 C5’ – 120.69 129.98 114.44 129.00 16 C6’ – 130.48 120.09 129.43 129.57 17 α 25.97 26.95 26.10 25.03 26.09 18 For Peer Review 19 β – – – – – 20 γ – – – – –
21 Cc – 55.65 55.65 55.24 21.61 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 34 of 34
1 2 13 3 Table 5. 100 MHz C NMR spectra of HCl salts in CDCl 3. 4 Carbon TCE ( 1b ) 2 MeO PCE ( 2b ) 3 MeO PCE ( 3b ) 4 MeO PCE ( 4b ) 3 Me PCE ( 5b ) 5 C1 62.60 64.81 64.30 63.94 64.15 6 C 34.93 32.54 33.44 33.39 33.33 7 2,6 8 C3,5 22.36 22.76 22.30 22.23 22.29 9 C4 24.71 24.92 25.05 25.13 25.09
10 C1’ 139.98 123.96 137.01 126.86 135.19 11 C 128.88 157.06 113.26 129.72 128.83 12 2’ 13 C3’ 127.86 111.94 160.42 114.43 138.99 14 C4’ 126.98 130.50 115.01 159.61 125.31 15 C5’ – 121.23 129.94 114.43 128.98 16 C6’ – 130.37 120.41 129.72 129.47 17 α 36.73 37.25 36.79 36.58 36.76 18 For Peer Review 19 β 11.95 12.84 12.07 11.98 12.01 20 γ – – – – –
21 Cc – 55.57 55.72 55.23 21.66 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Page 35 of 34 Drug Testing and Analysis
1 2 13 3 Table 6. 100 MHz C NMR spectra of HCl salts in CDCl 3. 4 Carbon TCPr ( 1c ) 2-MeO-PCPr ( 2c ) 3-MeO-PCPr ( 3c ) 4-MeO-PCPr ( 4c ) 3-Me-PCPr ( 5c ) 5 C1 62.73 64.84 64.42 64.05 64.26 6 C 34.89 32.32 33.21 33.36 33.29 7 2,6 8 C3,5 22.40 22.79 22.34 22.22 22.32 9 C4 24.71 24.86 25.05 25.12 25.08
10 C1’ 139.98 123.92 137.02 126.88 135.20 11 C 128.91 156.81 113.23 129.74 128.85 12 2’ 13 C3’ 127.85 111.93 160.45 114.41 139.00 14 C4’ 126.98 130.57 115.05 159.59 125.33 15 C5’ – 121.36 129.92 114.41 128.94 16 C6’ – 130.41 120.44 129.74 129.48 17 α 43.29 43.64 43.35 43.17 43.31 18 For Peer Review 19 β 19.95 20.30 20.07 19.97 20.00 20 γ 11.49 11.05 11.45 11.45 11.44
21 Cc – 55.53 55.74 55.22 21.67 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta